Dimerization and phosphorylation of Lutheran/basal cell adhesion molecule are critical for its function in cell migration on laminin.

Tumor cell migration depends on the interactions of adhesion proteins with the extracellular matrix. Lutheran/basal cell adhesion molecule (Lu/BCAM) promotes tumor cell migration by binding to laminin α5 chain, a subunit of laminins 511 and 521. Lu/BCAM is a type I transmembrane protein with a cytoplasmic domain of 59 (Lu) or 19 (Lu(v13)) amino acids. Here, using an array of techniques, including site-directed mutagenesis, immunoblotting, FRET, and proximity-ligation assays, we show that both Lu and Lu(v13) form homodimers at the cell surface of epithelial cancer cells. We mapped two small-XXX-small motifs in the transmembrane domain as potential sites for monomers docking and identified three cysteines in the cytoplasmic domain as being critical for covalently stabilizing dimers. We further found that Lu dimerization and phosphorylation of its cytoplasmic domain were concomitantly needed to promote cell migration. We conclude that Lu is the critical isoform supporting tumor cell migration on laminin 521 and that the Lu:Lu(v13) ratio at the cell surface may control the balance between cellular firm adhesion and migration.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

INTRODUCTION: KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. STUDY DESIGN AND METHODS: In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. RESULTS: Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. CONCLUSION: Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

A new method for quantitative analysis of cell surface glycoproteome.

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface-capturing strategy where the glycopeptides were submitted to LC-MS/MS analysis directly for identification of glycoproteins and the non-glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.

[Study on the gene polymorphism of Auberger antigens in Chinese population].

OBJECTIVE: To study the gene polymorphism of the Auberger antigens in Lutheran blood group system in Chinese population and establish a stable, accurate molecular method detecting Auberger antigens. METHODS: Peripheral blood samples from 162 randomly collected and unrelated volunteer blood donors were directly sequenced for the exon 12 at the gene locus of Auberger antigens. PCR products with novel nucleotide were further investigated by restriction fragment length polymorphism (RFLP) analysis. RESULTS: Auberger genotypes in the 162 Chinese individuals were obtained: Au(a+ b- )(nt1615A) was found in 119 individuals, Au(a+ b+ ) (nt1615A/G) in 40 individuals and Au(a- b+ ) (nt1615G) in 3 individuals. The allele frequencies of the Au(a) and Au(b) were 0.8580 and 0.1420, respectively. An individual with homozygous Au(a) genotype had a nucleotide mutation (1595 G to T). The mutation was confirmed by digesting the DNA with Hha I. CONCLUSION: The distribution of gene polymorphism of Auberger antigens in a Chinese population was investigated and obtained. And a molecular method determining the Auberger antigen was established. A novel Lutheran allele was deposited in GenBank (accession number EU260043).

Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin.

The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the alpha spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of alpha-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

Molecular bases of the antigens of the Lutheran blood group system.

BACKGROUND: Lutheran is a complex blood group system consisting of 18 identified antigens. There are four pairs of allelic antigens, whereas others are independently expressed antigens of a high frequency. Lutheran antigens are carried by the Lutheran glycoproteins, which are a product of a single gene LU. STUDY DESIGN AND METHODS: Genomic DNA from 21 individuals of 12 Lutheran phenotypes was used for PCR amplification of selected LU exons that were directly sequenced and compared to control DNA of a common Lutheran phenotype. RESULTS: Lutheran phenotypes were mostly caused by single-nucleotide polymorphisms within LU, resulting in single amino acid changes. The following mutations were observed: in LU:-4, G524A, Arg175Gln; in LU:-5, G326A, Arg109His; in LU:-6,9, C824T, Ser275Phe; in LU:-8,14, T611A, Met204Lys; in LU:-13, three point mutations (C1340T, Ser447Leu, C1671T silent mutation for Ser557 and A1742T, Gln581Leu); in LU:-16, C679T, Arg227Cys; in LU:-17, G340A, Glu114Lys; and in LU:-20, C905T, Thr302Met. Two LU:-12 samples had differing results: one individual had a deletion 99GCGCTT, Arg34 and Leu35, whereas the second LU:-12 sample had a point mutation G419A, Arg140Gln. CONCLUSION: The results revealed the genetic background of 11 Lutheran antigens and suggested their placement on the Lutheran glycoprotein.

Characterization of the laminin binding domains of the Lutheran blood group glycoprotein.

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding.

Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease.

Erythroid cell adhesion molecules Lutheran and LW in health and disease.

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.